The invention relates to synthetic peptides having diuretic, natriuretic, and/or vasodilatory activity in mammals. The peptides of this invention are related to atrial natriuretic peptide (ANP) but exhibit decreased binding affinity to the natriuretic peptide clearance receptor (NPR-C) and improved stability toward peptidases (xe2x80x9cenkephalinasexe2x80x9d) known to hydrolyze natriuretic peptides.
Maintenance of normal extracellular fluid volume depends primarily on the excretion of sodium (natriuresis) and water (diuresis) by the kidneys. These are, in turn, primarily determined by (1) the rate at which plasma is filtered at the glomerulus (glomerular filtration rate, or GFR) and (2) the degree to which sodium is actively reabsorbed along the renal tubule (with water presumably following passively). Sodium reabsorbtion is regulated, in part, by the adrenal steroid hormone aldosterone, in part by blood pressure, hematocrit and plasma viscosity and in part by various atrial natriuretic factors (ANF""s) or hormones (deBold, A. J. et al., Life Sciences 28:89-94 [1981]; Garcia R., Experientia 38:1071-73 [1982]; Currie, M. S. et al. Science 221:71-73 [1983]; Flynn T. G. et al., Biochem. Biophys. Res. Commun. 117:859-865 [1983]; Currie, M. G. et al., Science 223:67-69 [1984]; and Kanagawa, K. et al., Biochem. Biophys. Res. Commun. 118:131-139 [1984]).
Atrial natriuretic factors are released as a result of sensors in the atrium responsible for detecting extracellular fluid volume. It is believed that an increase in extracellular fluid volume is detected by these sensors as the atrium stretches to accommodate the increased venous return volume. In response to this stimulus ANF is released into the blood stream where it is transported to the kidney. Binding of ANF to a specific natriuretic peptide receptor (hNPR-A) in the kidney causes inhibition of sodium reabsorbtion along the renal tubule decreasing the reabsorption of water and lowering the extracellular fluid volume.
ANF is also known to be a hypotensive hormone that antagonizes various hypertensive systems including; stimulation of vasodialation, inhibition of aldosterone secretion from the adrenal gland, renin secretion from the kidney, and the renin-angiotensin II system.
It is known that the serum half-life of ANF""s and related peptides (see FIG. 1) that act as hypotensive regulators is relatively short (Crozier, I. G. et al., The Lancet II 1242-1245 [1986]) and that these peptides are removed from the blood stream by several mechanisms (FIG. 2). In addition to glomerular filtration two other distinct pathways have been identified which appear to significantly contribute to ANF clearance.
The first of these pathways involves receptor mediated ANF clearance. This pathway is reported to have sufficient capacity to account for about 70-80% of total ANF clearance from the blood stream (Maack, T., et al, Science 238:675-678 [1987], EPO Publication No. 233,143). The human natriuretic peptide receptor (hNPR-C) responsible for this clearance is present in many tissues, especially kidney (Luft, F. C. et al., J. Pharmacol. Exp. Ther. 236:416-418 [1986]), and promiscuously (Bovy, P. R., Med. Res. Rev. 10:1156 [1990]) binds various human natriuretic peptides including hANP, hBNP, and hCNP (FIG. 3). Various synthetic peptides, especially linear peptides (Olins, G. M., et al., J. Biol. Chem. 263:10989-10993 [1988]), capable of binding the hNPR-C have been described in the patent literature to enhance the natriuretic, diuretic and vasodilatation activity of endogenous ANF. Therapeutic use of these clearance receptor inhibitors presumably elevates the concentration and thus activity of all hormone peptides cleared by hNPR-C.
A second nonsaturatable clearance pathway also operates to remove serum ANF and is believed to involve the activity of a peptidase, neutral endopeptidase 24.11 (EC 3.4.24.11), also known as xe2x80x9cenkephalinasexe2x80x9d and referred to herein as NEP (Stevenson, S. L., et al., Biochem. 243L183-187 [1987]; Olins, G. M., et al., Biochim Biophys Acta 901:97-100 [1987]; Koehn, J. A. et al., J. Biol. Chem. 262:11623-11627 [1987]; Yandle, T., et al., Biochem Biophys Res. Commun. 146:832-839 [1987]). NEP is present in relatively high amounts in the kidney (Sonnenberg, J. L. et al., Peptides 9:173-180 [1988]) and is known to hydrolyze the Cys7-Phe8 amide bond of ANF""s (Tamburine, P. P., et al., J. Pharm. Exp. Ther. 251:956-961 [1989]).
It has been observed that inhibitors of NEP, such as thiophan, potentiate the biological responses of administered ANP (Fennell, S. A., et al., FASEB J 2:A936 [1988]); Seymour A. A. et al., ibid; Trapani, A. J., et al., ibid; McMartin, C., et al., ibid; Simmerman, M. B. et al. ibid A937). However, administration of nonpeptidyl inhibitors of this pathway, like thiorphan, has the disadvantage that the cerebral NEP or endopeptidase 24.11 (xe2x80x9cenkephalinasexe2x80x9d) will also be inhibited because thiorphan is capable of crossing the blood-brain barrier (Bourgoin, S. et al., J. Pharm. Exp. Ther. 238:360-366 (1986). In addition to the use of thiorphan, a variety of other strategies for the inhibition of NEP have been described. These strategies include the use of a metal binding substituent appropriately spaced from the aromatic Phe8 moiety of ANF. Roques., E. P., et al., Nature 288:286-288 (1980); see also Gordon, E. M., et al., Life Sci 33 (Supplement 1): 113-116 (1983); Mumford, R. M., et al., Biochem Biophys. Res. Comm. 109:1303-1309 (1982); Fournie-Zaluski, M. C., et al., J. Med. Chem. 26:60-65 (1983); Waksman, G., et al., Biochem. Biophys. Res. Comm. 131:262-268 (1985); U.S. Pat. No. 5,248,764. Other strategies also include substitution of unnatural residues for Phe8 such as cyclohexylamine (Fed. Proc., 45:657 [1986]; U.S. Pat. Nos. 5,106,834 and 4,861,755) or N-alkylated amino acids like N-Me-Phe (Nutt et al., EPA 0 465 097). Introduction of D amino acids such as D-Cys or D-Ala has been described (Nutt R. and Veber, D. F. Endocrin. Metab, Clin. N. Am., 16:19-41 [1988]; U.S. Pat. No. 4,816,443) and replacement of amide bonds is described generally be Lewicki et al., U.S. Pat. No. 4,935,492 (see also U.S. Pat. No. 5,095,004).
Because of the obvious therapeutic potential of natriuretic peptides and variants thereof in the treatment of congestive heart failure, hypertension, acute kidney failure etc., numerous synthetic ANF""s have been prepared that mimic the biological activity of wild-type ANF but are reported to have improved stability, potency, or duration of action when compared to wild-type ANF. Many of these synthetic ANF""s are disclosed in the following U.S. Pat. Nos.: 4,496,544; 4,496,544; 4,609,725; 4,673,732; 4,716,147; 4,757,048; 4,764,504; 4,804,650; 4,816,443; 4,861,755; 4,935,492; 4,952,561; 5,057,495; 5,057,603; 5,091,366; 5,095,004; 5,106,834; 5,159,061; 5,204,328; and 5,212,286. In addition, various foriegn documents describing ANF analogs include: WO85/04870; WO85/04872; WO88/03537; WO88/06596; WO89/10935; WO89/05654; WO90/01940; WO90/14362; WO92/06998; EPA 0 323 740; EPA 0 356 124; EPA 0 291 999; EPA 0 350 318; EPA 0 497 368; EPA 0 385 476; and EPA 0 341 603. None of these publications disclose a hANF variant having human receptor specificity (i.e. high affinity for hNPR-A and low affinity for hNPR-C), nor do they disclose the substitutions to hANF(1-28) residues 9, 11, or 16 necessary to achieve this selectivity. Only WO88/03537 discloses a positively charged residue, D-Arg, at position 16 in a truncated form of ANF.
It is an object of this invention to provide novel compounds having biological activity like hANF(1-28) but with enhanced metabolic stability. It is another object of this invention to provide novel peptides or peptidomimetic compounds having potent vasodilatory, natriuretic, and hypotensive activity. It is a further object to provide novel compounds having enhanced specificity for the hNPR-A in relation to other hNPR""s, especially hNPR-B and hNPR-C. These and other objects of the invention will be apparent from the following specification.
The objectives of this invention have been met by providing novel compounds having potent vasodilatory, natriuretic, and/or hypotensive activity and having enhanced specificity for hNPR-A in relation to other hNPR""s, especially hNPR-B and hNPR-C. The compounds of this invention generally have a decreased binding affinity for the human clearance receptor, HNPR-C, compared to the wild-type protein hANF(1-28). These compounds preferably also have an equal or higher affinity for the human A receptor, hNPR-A, compared to the wild-type protein hANF(1-28). These biological characteristics are normally achieved by providing one or more of the following substitutions in wild-type hANF(1-28) or mutants thereof; G9T, G9a, G9R, R11S, R11D, G16R, G16K, G16Orn and G16Har. Additionally these compounds have an increased half-life relative to wild-type hANF(1-28) when incubated with neutral endopeptidase 11.24 (NEP). The increased half-life is preferably achieved by; adding the urodilatin amino terminus tetrapeptide sequence, adding a (Ser)4 sequence to the amino terminus, modifying the Cys7-Phe8 amide bond with an amide isostere, N-alkylating Cys7, Phe8, Ala17, Ile15 or Phe26, or inserting a D-amino acid between Cys7 and Phe8. A preferred compound having hNPR-A selectivity is represented by Formula (I) 
where
X is selected from
H, C1-C6alkanoyl, Ser-Pro-Lys-, (Ser)4- and Thr-Ala-Pro-Arg- (SEQ ID NO: 1);
AA1 is absent or is selected from
Ser, Met, Gly, Asp, Ala, Tyr and His;
AA2 is absent or is selected from
Leu, Asp, Met, Val, Ser, Ala, Phe, Pro and Lys;
AA3 is absent or is selected from
Cys, Glu, Ser, Gln, His, Gly, Arg and Asp;
AA4 is absent or is selected from
Arg, Gly, Ala, Asp, Met, Leu, Tyr and Pro;
AA5 is absent or is selected from
Ser, Cys and Asp;
AA6 is absent or is selected from
Ser, Gly and Glu;
AA7 is selected from
Cys, N-methyl-Cys, D-Cys and Pen;
AA8 is selected from
Phe, N-methyl-Phe, Trimethylphenyl-Ala, halo(F, Cl, Br and I)phenyl-Ala, Trifluoromethylphenyl-Ala, Tyr, O-methyl-Tyr, Cha, xcex2-napthyl-Ala, xcex1-napthyl-Ala, biphenyl-Ala, diphenyl-Ala, D-Ala, dibenzyl-Ala, florenyl-Ala, adamantyl-Ala, and xcex2-napthyloxy-Ala;
AA9 is selected from
Gly, Arg, Thr, Val, Asp, Ala, D-Ala, Pro and Glu;
AA10 is selected from
Gly, Arg, Ser, Ala, His, Pro and Lys;
AA11 is selected from
Arg, Lys, N-methyl-Arg, Asp, Ser and Pro;
AA12 is selected from
Met, Ile, D-Leu, Nle and Leu;
AA13 is selected from
Asp and Glu;
AA14 is selected from
Arg, N-methyl-Arg, Pro and Ser;
AA15 is selected from
Ile, N-methyl-Ile and Leu;
AA16 is selected from
Gly, Tyr, Phe, Ser, Tyr, Pro and a positively charged amino acid residue is selected from
Orn, Har, Lys, p-amidinophenyl-Ala and Arg;
AA17 is selected from
Ala, Ser, N-methyl-Ala, Glu and Pro;
AA18 is selected from
Gln, Ser, Ala and homo-Cys;
AA19 is selected from
Ser;
AA20 is selected from
Gly and Ala;
AA21 is selected from
Leu;
AA22 is selected from
Gly and Ala;
AA23 is selected from
Cys;
AA24 is selected from
Asn;
AA25 is selected from
Ser and Val;
AA26 is selected from
Phe, D Phe, N-methyl-Phe and Leu;
AA27 is selected from
Arg, Orn, Har, Lys, p-amidinophenyl-Ala and Arg-Arg;
AA28 is selected from
Tyr and homoCys; and
Z is selected from
OH and NR1R2 where R1 and R2 are independently selected from
H, C1-C6alkyl, C6-C12aryl and C6-C12aryl-C1-C6alkyl;
and where
the amide bond (xe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94) bonding residues AA7 and AA8 may optionally be replaced with an amide isostere selected from the group
xe2x80x94CH2xe2x80x94NHxe2x80x94,
xe2x80x94CH2xe2x80x94Sxe2x80x94,
xe2x80x94CH2xe2x80x94S(O)nxe2x80x94, where n is 1 or 2,
xe2x80x94CH2xe2x80x94CH2xe2x80x94,
xe2x80x94CHxe2x95x90CHxe2x80x94 (E or Z),
xe2x80x94C(xe2x95x90O)xe2x80x94CH2xe2x80x94,
xe2x80x94CH(CN)xe2x80x94NHxe2x80x94,
xe2x80x94C(OH)xe2x80x94CH2xe2x80x94, and
xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94
provided that one of AA9, AA11, and AA16 is selected according to the following scheme
AA9 is Arg, Thr or Glu;
AA11 is Asp, Ser or Pro; and
AA16 is a positively charged amino acid residue selected from
Arg, homoArg (Har), Lys, Orn, and p-amidinophenyl-Ala; and
pharmaceutically acceptable salts thereof.
Preferably AA16 in the compound of Formula I is a positively charged amino acid residue selected from; Orn, Har, Lys, p-amidinophenyl-Ala or Arg, and most preferably Arg. Also preferably and independently, AA9 is selected from Arg, Thr, or Glu, and AA11 is selected from Asp, Ser, or Pro. Optionally, AA3 is Asp, X is Thr-Ala-Pro-Arg- (SEQ ID NO: 1) and/or the compound may contain a second disulfide (or equivalent) bond.
Optionally, compounds of Formula I may have a D-amino acid residue inserted between AA7 and AA8.
Most preferred hANF variants of this invention include:
hANF(1-28) G9T, R11S, M12I;
hANF(1-28) G9R, R11D, M12I, G16R;
hANF(1-28) G9T, R11S;
hANF(1-28) G9E, G10R, R11P;
hANF(1-28) G10K, R11S;
hANF(1-28) M12I, G16R;
hANF(1-28) G9T, R11S, G16R, Q18P;
hANF(TAPR 1-28) M12I, G16R;
hANF(1-28) M12I, R14S, G16R;
hANF(1-28) G9E, G10R, R11P, M12I, G16R;
hANF(1-28) R3D, G9T, R11S, M12L, R14S, G16R;
hANF(1-28) G9T, R11S, M12I, G16R, inserting D-Ser between residues 7 and 8;
hANF(1-28) G9T, R11S, M12I, G16R, inserting D-Ala between residues 7 and 8;
hANF(TAPR1-28) G13T, R15S, M16I, G20R;
hANF(1-28) F8Cha(1-cyclohexylalanine), G9T, R11S, M12I, G16R;
Mpa(mercaptopropionic acid), F, D-Thr, hANF(10-28), G9T, R11S, M12I, G16R;
Mpa (mercaptopropionic acid), F, D-Ala, hANF(10-28), G9T, R11S, M12I, G16R;
hANF(1-28) C7(N-MeCys), G9T, R11S, M12I, G16R;
hANF(1-28) F8(N-MePhe), G9T, R11S, M12I, G16R;
hANF(1-28) C7F8 replaced by C("psgr"CH2NH)F*, G9T, R11S, M12I, G16R;
hANF(1-28) G9(d-Ala), R11D, M12I, G16R;
hANF(1-28) F8Phg(l or d phenyl glycine), G9T, R11S, M12I, G16R;
hANF(1-28) R3D, G9T, R11S, M12L, R14S, G16R, Q18C*, Y28C*;
hANF(1-28) M12I, G16R, A17E, Q18A;
hANF(1-28), C7F8 replaced by C("psgr"CH2NH)F, G9T, R11S, M12I, G16R, F26(N-Me-Phe); and
hANF(Ac-1-28), C7F8 replaced by C("psgr"CH2NH)F, G9T, R11S, M12I, G16R.
The invention further comprises a pharmaceutical composition including a pharmaceutically acceptable excipient and any of the compounds described above. This composition is used in a method for inducing naturesis, diuresis, vasodilation or to modulate the renin-angiotensin II and aldosterone systems.